CN111484969A

CN111484969A - Application of hair follicle stem cell source exosome in promoting hair follicle stem cell proliferation and differentiation to hair follicle cells

Info

Publication number: CN111484969A
Application number: CN202010378196.3A
Authority: CN
Inventors: 高峰; 刘迎春; 陈钰萌; 彭耀
Original assignee: Inner Mongolia Agricultural University
Current assignee: Inner Mongolia Agricultural University
Priority date: 2020-05-07
Filing date: 2020-05-07
Publication date: 2020-08-04
Anticipated expiration: 2040-05-07
Also published as: CN111484969B

Abstract

The invention discloses application of a hair follicle stem cell source exosome in promoting hair follicle stem cell proliferation and differentiation to hair follicle cells. The invention discovers that exosomes derived from hair follicle stem cells can enter receptor cells and can promote the proliferation of the hair follicle stem cells and hair papilla cells; wherein, the melatonin-stimulated exosome derived from the hair follicle stem cells has a more obvious effect of promoting the proliferation of the hair follicle stem cells. The invention further discovers that the exosome derived from the hair follicle stem cells can promote the migration of the hair follicle stem cells and hair papilla cells, and the effect of promoting the migration of the exosome derived from the hair follicle stem cells stimulated by the melatonin is more obvious. The exosome derived from the hair follicle stem cells can promote the expression of genes related to hair follicle development of the hair follicle stem cells and hair papilla cells, so that the differentiation of the hair follicle stem cells to the hair follicle cells is promoted, and the effect of promoting the proliferation and differentiation of the hair follicle stem cells by the exosome derived from the hair follicle stem cells stimulated by melatonin is more obvious.

Description

Application of hair follicle stem cell source exosome in promoting hair follicle stem cell proliferation and differentiation to hair follicle cells

Technical Field

The invention relates to a new application of a hair follicle stem cell source exosome, in particular to an application of the hair follicle stem cell source exosome in promoting hair follicle stem cell proliferation and promoting hair follicle stem cell differentiation to hair follicle cells, belonging to the field of new applications of the hair follicle stem cell source exosome.

Background

Goats (Capra hircus), also known as summer, black or sheep, are, like sheep, among the first domesticated livestock to be domesticated by humans. It is classified in phylum vertebrata, class Mammalia, order Artiodactyla, family Bovidae, subfamily Caprae, and genus caprine. The goat breeds are classified into three types, i.e., dairy type, meat type and cashmere type, according to their economic uses.

Cashmere is one of the best and softest fibers produced by animals, and is used exclusively for the production of luxury textile products. Most cashmere is produced in China, Mongolia, Iran and other countries, and the cashmere production makes important contribution to economy. China is the world's largest cashmere producing country, accounting for 50% of world cashmere production, of which about 30% are from inner mongolian cashmere goats. The inner Mongolia cashmere goats are mostly distributed in the west of the inner Mongolia autonomous region, and have strong physique. The cashmere produced by the inner Mongolia cashmere goat has good quality and high yield, and comprises ideal color (white), brightness and elasticity. The growth of inner mongolian cashmere presents seasonal variations, i.e. variations of the natural photoperiod, cashmere growth usually starts from 7 months and then stops in 3 months, wool falls off at the bottom of 4 months.

Mammalian epidermis comprises several self-renewing compartments, containing multiple types of cells. Including epidermal stem cells, keratinocyte-progenitor cells from hair follicles, melanocyte progenitor cells, cutaneous neural stem cells, exocrine gland stem cells, skin-derived precursors (SKPs) located at the dermal papilla, hair follicle stem cells (Hairfollicula stem cells) in which cells located in the follicular bulge region express nestin (Yu H, Fang D, Kumar SM, et al. isolation of a genetic perturbation of multiple adult cells from human hair folliculas. the American journel of Pathology.2006 Jun; 168: 1879-88.). Stem cells of the follicle located in the follicular bulge region produce follicle structures at each anagen phase of the hair (Paus R, Cotsaris G. biology of hair follicals. the New England journal of media.1999 Aug 12; 341(7): 491-7.). Like most adult stem cells, hair follicle stem cells have slow periodicity, self-renewal, in vitro proliferation and multipotent differentiation potential, and are primitive cells in hair follicles. The periodic self-renewal and growth of Hair depends on the periodic proliferation and differentiation of the Hair Follicle Stem cells (Hoffman RM. introduction to Hair-folliculle-associated pluratent Stem cells. methods in molecular biology (Clifton, NJ). 2016; 1453: 1-5.).

The hair follicle stem cells have a multipotent differentiation potential and can differentiate into various types of cells, such as nerve cells, adipocytes, glial cells, keratinocytes, smooth muscle cells, cardiomyocytes, melanocytes, osteocytes and chondrocytes, etc. (He N, Dong Z, Zhu B, Nuo M, Bou S, L iu D.expression of pluripotency marker in arms and hair dye cells in vitro cellular & developmental biology animal 2016 Aug; 52(7):782-8.) furthermore, the hair follicle stem cells can regenerate epidermis. the differentiation potential of the hair follicle stem cells is greatest in the upper part of the hair follicle, which can produce a large number of these stem cells (Amoh Y, Mii S, Aki R, et al. multipotent nerve-expressing M of cell culture of cement tissue of cell 23. 13. 9. fig.: 23. 7. cell culture of cement tissue culture of cell culture of the genus of culture of cell 13. 7. fig.: 13. 7. the present invention can be applied to the present invention.

Hair papilla cells (DPCs) are a special fibroblast, in the lowermost part of the hair follicle the dermal papilla is identified as a permanent and stable population of specialized fibroblasts that first appear as aggregates of cells that interact with the epidermis to ensure hair follicle development, believed to provide signals that coordinate hair growth (Oliver RF, Jahoda. Dermal-epidermal interactions. Clinics in dermatology.1988 Oct-Dec; 6(4):74-82.) have many important roles in the regulation of hair growth. therefore, in vitro models of these cells are widely used to study the molecular mechanisms of hair follicle induction, growth and maintenance (Topozicell H, L ogN J, Williams G, Higgins CA. for the same and 3d differentiation of hair follicle cells) and to provide potential therapeutic potential for hair follicle repair cells of hair follicle cells of the lineage, especially T7-follicle tissue type 27, and T7 tissue repair cell series for hair follicle development, and T7. 1. A. host for the same kind of hair follicle tissue repair and tissue repair of hair follicles, especially for hair follicle tissue repair of hair follicle tissue growth

The first observation of exosomes (exosomes) by Pan and Johnstone before 30 years, and the maturation of reticulocyte erythroblasts, indicated by "vesicles", later called "exosomes", shed from cultured monolayer cells and retained transferrin receptor and many membrane-associated proteins until ten years after the "vesicles" phenomenon was found, Raposo (Raposo G, Nijman HW, Stoorvogel W, et al. B lymphocyte induced antigen-expressing vector, the present Journal of extracellular medium, 1996; Mar 1161-72.). until the "vesicles" were found, all found to be able to isolate from virus infected B DJ cells, have had many extracellular antigens and extracellular immune responses, extracellular responses, expressed by lymphocytes, extracellular vesicles, extracellular antigens, secreted by saliva cells, extracellular vesicles, extracellular antigens, secreted by saliva, extracellular vesicles, saliva, urine, saliva, urine, saliva, urine, saliva, urine, saliva, urine, saliva, urine, saliva, urine, saliva, urine, saliva, urine, saliva, urine, saliva, urine, saliva, urine, saliva, urine, saliva, urine, saliva, etc. 2, etc. 1, saliva, etc. 1, etc. the like, etc. 2, etc. the like, etc. the cells, etc. are shown in which were shown in the cells, the cells.

Exosomes are thought of as distinct vesicle populations, different in size from the microvesicles. Exosomes are defined as vesicles in the 30-100nm range, whereas microvesicles are defined as vesicles in the 100-1000nm range. However, despite this apparent distinction, the terms "exosomes" and "microvesicles" are used interchangeably in many published reports (van der Pol E, Boing AN, Harrison P, Sturk A, Nieuwland R.Classification, functions, and clinical reservability of extracellular vehicles. pharmacological reviews.2012 Jul; 64(3): 676-) 705.).

Biogenesis of exosomes consists of two steps, i.e. inward budding of endosomal membrane vesicles and their release into the structure of Multivesicles (MVBs). Early exosomes are formed when the cell membrane containing the surface protein undergoes endocytosis. Early exosomes recognize, classify, and select exosome-containing proteins under the action of vesicle sortilins (e.g., transport essential endosome complexes (ESCRTs)), thereby forming multivesicles; or forming multivesicular bodies with the aid of ceramides, and the process is divided into an ESCR-dependent pathway and an ESCR-independent pathway according to whether participation of ESCR is required. Briefly, as intraluminal vesicles accumulate, MVB formation occurs from early endosome to late endosome maturation. After maturation, MVBs are directed to fusion with lysosomes, where their vesicles will undergo lysosomal or plasma membrane degradation, where their contents will be released into the extracellular space.

Exosomes carry a unique protein, lipid and RNA, which can be distinct from the original cell. Exosomes provide guarantee for proteins, lipids, and RNA contained inside them, protect them from degradation by enzymes, and enter cells by endocytosis. Because exosomes are from endosomes, they contain proteins important for trafficking and fusion, such as annexins; there are tetraspanin proteins involved in cell targeting; there are biogenic proteins involved in their passage from MVB, such as Alix and TSG 101; (ii) a platelet-derived growth factor receptor; there are lysosome-associated membrane proteins, Heat Shock Proteins (HSPs), lipid-associated proteins, and the like. High throughput proteomic analysis of exosomes revealed the presence of extracellular matrix and cell surface proteins, such as collagen, integrins and galectins; cell surface receptors such as platelet derived growth factor receptor B and Epidermal Growth Factor Receptor (EGFR); and signaling molecules and intracellular cytoskeletal components plus metabolic enzymes and G proteins. Research shows that the protein contained in the exosome has no direct relation with the cell from which the exosome is derived, and the protein types of most exosomes are consistent. Therefore, some typical proteins are considered as exosome surface markers, which can be used to identify exosomes in general. Including four-molecule cross-linked family members (CD9, CD63, CD81), tumor susceptibility gene 101(Tsg101) proteins and Heat Shock Proteins (HSPs). Exosomes may bind to receptors or ligands on the membrane surface of the cell membrane of a recipient cell, and may internalize to activate intracellular signaling pathways, thereby altering its cellular phenotype.

Exosomes may carry functional mRNA and miRNA that are transferred between cells (Valadi H, Ekstrom K, bossisos a, Sjostrand M, L ee JJ, &lttt transfer = L "&gtt L &ttt/t gtt otl otval jo. exosome-mediated transfer of mrnas and microRNAs a novel mechanism of genetic exchange between genomic and tissue, naturecell biology.2007 Jun; 9(6):654-9.), exosomes RNA content is a subset of cellular RNA, in some cases may be completely different or tissue specific, whatever the source cell is, due to exosomes specifically targeting mvb during biogenesis, tumor-releasing microvesins containing microrna, genomic DNA and genomic DNA, unique genes, genomic DNA, transgene, genomic DNA, unique genes, genomic DNA, unique sequences, genomic DNA, unique sequences, antisense.

A large number of proteins and receptors (such as CD63, CD81, CD9 and HSPs) exist on the surface of an exosome membrane, and can be identified by a Western blot method through specific binding of the proteins and antibodies. Morphological observation can also be performed by transmission electron microscopy, when observed under transmission electron microscopy, exosomes are typically saucer-shaped, 30-100nm in diameter. Further identification of exosomes requires the assistance of proteomics analysis, Fluorescence Activated Cell Sorting (FACS), Nanoparticle Tracing (NTA) and other means.

To date, there has been no report on the function or activity of exosomes derived from hair follicle stem cells.

Disclosure of Invention

One of the objects of the present invention is to provide a method for promoting secretion of exosomes from hair follicle stem cells.

Another objective of the invention is to provide a method for promoting the proliferation of hair follicle stem cells and the differentiation of the hair follicle stem cells into hair follicle cells.

It is another object of the present invention to provide a method for promoting proliferation of dermal papilla cells.

The above object of the present invention is achieved by the following technical solutions:

on the basis, the invention further discovers that the hair follicle stem cells can secrete exosomes, and the stimulation of the hair follicle stem cells by the melatonin can promote the hair follicle stem cells to secrete the exosomes, wherein the stimulation of the hair follicle stem cells by 100-dose 700pg/m L melatonin can promote the hair follicle stem cells to secrete the exosomes, and particularly the stimulation of the hair follicle stem cells by 500-dose m L melatonin can most obviously promote the hair follicle stem cells to secrete the exosomes.

The invention further discovers that the exosome from the hair follicle stem cell promotes the proliferation, migration and development expression of the related genes of the hair follicle stem cell and the hair papilla cell, and can promote the hair follicle stem cell to be differentiated to the hair follicle cell; in addition, the exosome-promoting effect secreted after stimulating the hair follicle stem cells with melatonin was more pronounced and increased with increasing concentration gradient.

The experiments show that after exosomes are added into hair follicle stem cells, the proliferation of the hair follicle stem cells is increased, and the proliferation is obviously increased along with the increase of the concentration gradient, compared with exosomes CG-Exo from untreated hair follicle stem cells and exosomes L uz-Exo and 4-P-Exo added with melatonin (receptor blocking), exosomes MT-Exo from melatonin-treated hair follicle stem cells can obviously promote the increase of the hair follicle stem cells, so the exosomes from the hair follicle stem cells can promote the proliferation of the hair follicle stem cells, and the exosomes from the hair follicle stem cells treated with the melatonin can obviously promote the proliferation of the hair follicle stem cells.

Experiments further discover that the exosome from the hair follicle stem cells can promote the hair papilla cells to proliferate, and the exosome from the hair follicle stem cells treated by the melatonin has a more obvious effect of promoting the hair papilla cells to proliferate.

Therefore, the invention provides a method for promoting secretion of exosomes from hair follicle stem cells, which comprises stimulating the hair follicle stem cells with melatonin to promote secretion of exosomes from the hair follicle stem cells, wherein the concentration of the melatonin added is preferably 100-700pg/m L, and more preferably 500pg/m L.

The invention also provides a method for promoting the differentiation of hair follicle stem cells to hair follicle cells, which comprises the following steps: adding exosomes secreted by the hair follicle stem cells into the hair follicle stem cells; wherein, when preparing the exosome secreted by the hair follicle stem cells, melatonin is added into the hair follicle stem cells to promote the exosome secreted by the hair follicle stem cells.

The invention also provides a method for promoting the proliferation of hair follicle stem cells, which comprises the following steps: adding exosomes secreted by the hair follicle stem cells into the hair follicle stem cells to promote the hair follicle stem cells to proliferate; wherein, when preparing the exosome secreted by the hair follicle stem cells, melatonin is added into the hair follicle stem cells to promote the exosome secreted by the hair follicle stem cells.

The present invention further provides a method of promoting proliferation of dermal papilla cells, comprising: adding exosome secreted by hair follicle stem cells into hair papilla cells to promote the proliferation of the hair papilla cells; wherein, when preparing the exosome secreted by the hair follicle stem cells, melatonin is added into the hair follicle stem cells to promote the exosome secreted by the hair follicle stem cells.

Detailed description of the invention

The invention firstly considers the influence of melatonin on the proliferation of hair follicle stem cells and the secretion of exosomes thereof, the experiment stimulates the hair follicle stem cells with the melatonin, extracts cell supernatant secretes thereof, and utilizes BCA protein for quantification, the results show that the quantification results are different, the quantity of exosomes of the origin of the melatonin-stimulated hair follicle stem cells is preliminarily determined, according to the MTT and BCA results, the early-stage data analysis of the experimental group is combined to judge that the proliferation of the hair follicle stem cells is the best when the melatonin is stimulated for 3d at 500pg/m L, the differentiation phenomenon does not occur, and the exosomes of the origin of the melatonin are accurately judged as the exosomes of the hair follicle stem cells, the invention finds that the melatonin with four concentrations of 100pg/m L, 300pg/m L, 500pg/m L and 700pg/m L has the promotion effect on the quantity of the exosomes of the hair follicle stem cells from in vitro cultured hair follicle stem cells, wherein, the 500pg/m L stimulation group can most obviously increase the quantity of the exosomes of the hair follicle stem cells when the hair follicle stem cells stimulate 3 d.

Under the culture of an exosome-free serum culture medium, four concentrations of melatonin of 100pg/m L, 300pg/m L, 500pg/m L and 700pg/m L have a promoting effect on the proliferation of hair follicle stem cells cultured in vitro, 500pg/m L is the optimal concentration for promoting the proliferation of the hair follicle stem cells, 100pg/m L, 300pg/m L, 500pg/m L and 700pg/m L have a promoting effect on the amount of exosomes derived from the hair follicle stem cells cultured in vitro, and the melatonin stimulation group of 500pg/m L is the exosome optimal concentration extraction time treatment group when 3d is stimulated.

The invention further considers the influence of melatonin on exosomes derived from hair follicle stem cells, HSP70, CD9 and CD81 are used as marker proteins for identifying exosomes, and experimental results show that the exosomes are positively expressed, β -actin is selected as an internal reference protein, and the expression levels of HSP70, CD9 and CD81 are obviously increased relative to the internal reference, so that the extraction and separation of exosomes in the experiment can be effectively used in subsequent experiments.

The hair follicle stem cell source exosomes MT-Exo after melatonin treatment can remarkably increase the hair follicle stem cell proliferation effect compared with the hair follicle stem cell source exosomes CG-Exo and secretion exosomes CG-Exo which are not treated, and the hair follicle stem cell proliferation promoting effect is remarkably similar to that of a hair follicle stem cell exosomal tissue treated by adding melatonin (receptor blocking) after the hair follicle stem cell treatment, so that the hair follicle stem cell proliferation promoting effect is remarkably increased, the hair follicle stem cell proliferation promoting effect is remarkably improved, and the hair follicle stem cell proliferation promoting effect is remarkably improved after the hair follicle stem cell exosomal tissue is added, so that the hair follicle stem cell proliferation promoting effect is remarkably improved after the hair follicle stem cell is added, the hair follicle stem cell proliferation promoting effect is remarkably similar to that of the hair follicle stem cell proliferation promoting effect after the hair follicle stem cell proliferation promoting effect is remarkably increased after the hair follicle stem cell proliferation promoting effect is obtained after the hair follicle stem cell is added, the hair follicle stem cell proliferation promoting effect is remarkably increased after the hair follicle stem cell proliferation promoting effect is remarkably increased by adding the melatonin (receptor blocking) exosomes CG-Exo after the hair follicle stem cell treated by adding the melatonin (receptor blocking) and adding the hair follicle stem cell.

The melatonin-treated hair follicle stem cell-derived exosome MT-Exo remarkably promotes the increase of hair follicle stem cells compared with the untreated hair follicle stem cell-derived exosome CG-Exo and the melatonin (receptor blocking) -treated exosomes L uz-Exo and 4-P-Exo, L uz-Exo and 4-P-Exo have small differences with CG-Exo, but for hair follicle cells, the different concentrations of the same exosome appear to be small differences within the first 2 days, particularly when the concentrations of 300 mu g/m L and 500 mu g/m L are added, but in general, the hair follicle stem cell-derived exosome can promote the proliferation of hair follicle papilla cells, and the effect of the melatonin-treated hair follicle stem cell-derived exosome on promoting the proliferation of hair follicle papilla cells is more remarkable.

The results of the transwell migration experiment show that compared with the control group, the group added with melatonin stimulation can promote migration of hair follicle stem cells, when the exosome derived from the hair follicle stem cells is added, the migration effect of the cells is rapidly increased, meanwhile, the exosome MT-Exo derived from the hair follicle stem cells after melatonin treatment can greatly promote the increase of the hair follicle stem cells compared with the exosome CG-Exo derived from the untreated hair follicle stem cells and the exosomes L uz-Exo and 4-P-Exo treated with melatonin (receptor blocking).

β -integrin and SOX9 are stem cell specific expression genes, after melatonin and melatonin treated exosomes are added to hair follicle stem cells, the expression level of the cells is gradually increased at 0-5d, and the expression level of the dry genes is reduced after 5 d.FGF 5 is a wool growth inhibition gene which mainly regulates the periodic development of hair follicles, and has expression in the growth process of hair follicles, and reaches the maximum expression level at 3d, and then the expression level is reduced. β -catenin has a great effect as an exogenous factor for regulating the differentiation of hair follicle stem cells as a key factor of Wnt signal channel.A highly activated β -catenin can lead hair follicle stem cells to differentiate towards hair follicle cells, and lacks or has low intensity activation of β -catenin to promote the differentiation into epidermal cells.A gene expression level is gradually increased at 0-3d, then the expression level is reduced, but after 7d, the gene expression level is continuously increased, possibly after stem cells are differentiated into hair follicle cells, a signal channel of 63-catenin continues to act as a KAP signal channel, and KAP7 and a marked by adding hair follicle stem cells which can not be rapidly expressed when the hair follicle stem cells are differentiated.

The invention discovers that exosomes derived from hair follicle stem cells can enter receptor cells; the hair follicle stem cell source exosome can promote the proliferation of hair follicle stem cells and hair papilla cells; wherein, the melatonin-stimulated exosome derived from the hair follicle stem cells has more obvious effect of promoting proliferation. The invention further discovers that the exosome derived from the hair follicle stem cells can promote the migration of the hair follicle stem cells and hair papilla cells, and the effect of promoting the migration of the exosome derived from the hair follicle stem cells stimulated by the melatonin is more obvious. The exosome derived from the hair follicle stem cells can promote the expression of genes related to hair follicle development of the hair follicle stem cells and hair papilla cells, can promote the differentiation of the hair follicle stem cells to the hair follicle cells, and has more obvious effect of promoting proliferation and differentiation of the exosome derived from the hair follicle stem cells stimulated by melatonin.

Drawings

FIG. 1 is a graph of the proliferative activity of hair follicle stem cells.

FIG. 2 protein standard curve.

Fig. 3 effect of melatonin on secretion of hair follicle stem cell exosomes.

FIG. 4 concentration of hair follicle stem cell-derived exosomes (μ g/m L) for each group.

FIG. 5 Transmission Electron microscopy of follicle stem cell-derived exosomes, A, untreated group follicle stem cell-derived exosomes (100 ×), B, untreated group follicle stem cell-derived exosomes (200 ×), C, treated group follicle stem cell-derived exosomes (100 ×), and D, treated group follicle stem cell-derived exosomes (200 ×).

FIG. 6 Western blot detection of exosome marker proteins derived from various groups of hair follicle stem cells.

FIG. 7 test results of the effect of hair follicle stem cell-derived exosomes on hair follicle stem cell proliferation.

FIG. 8 test results of the effect of hair follicle stem cell-derived exosomes on hair papilla cell proliferation.

FIG. 9 test results of the effect of hair follicle stem cell-derived exosomes on hair follicle stem cell migration.

FIG. 10 test results of the effect of hair follicle stem cell-derived exosomes on hair papilla cell migration.

FIG. 11 test results of the effect of exosomes derived from hair follicle stem cells on the regulation of the expression of KAP7 in the proliferation and differentiation of hair follicle stem cells.

FIG. 12 test results of the effect of hair follicle stem cell-derived exosomes on the regulation of hair follicle stem cell proliferation differentiation KAP8 expression.

Detailed Description

The invention is further described below in conjunction with specific embodiments, the advantages and features of which will become apparent from the description. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.

Experimental example 1 Effect of melatonin on proliferation of hair follicle stem cells and secretion of exosomes

1 test materials and reagents

1.1 Experimental materials

An isolated and purified inner Mongolian cashmere goat hair follicle stem cell line.

1.2 Experimental reagents

DMEM/F12, Fetal Bovine Serum (FBS) from Hyclone; DMSO, MTT from Coolaber; epidermal Growth Factor (EGF), Melatonin (Melatonin) was purchased from Sigma; Insulin-Transferrin-Selenium (ITS), 0.25% trypsin from Gibco; double antibody (PS) was purchased from solibao; total Exosome Isolation Reagent (from cell culture medium) was purchased from Invitrogen; RIPA lysate, BCA Protein Assay Kit, was purchased from Shanghai.

2. Experimental methods

2.1 thawing and Resuscitation of Hair follicle Stem cells

(1) The hair follicle stem cells of P3 generation (P4 generation after thawing) are taken out from liquid nitrogen, and are quickly put into a water bath at 37 ℃, and the mixture is clamped by tweezers and vigorously shaken until no obvious ice blocks exist, and then taken out. (2) Lightly rubbing with alcohol cotton ballWiping the tube mouth, then moving the tube mouth into an ultra-clean workbench, (3) sucking the cell fluid by a fluid moving gun, moving the tube mouth into a new centrifuge tube, centrifuging the tube at 1500r/min for 5min, (4) discarding the supernatant, re-suspending the stem cell culture fluid (containing DMEM/F12, 10% FBS, 1% PS, 20ng/m L EGF and 10 mu L/m L ITS), (5) adding the stem cell culture fluid into a 100mm culture dish, inoculating the cell suspension, placing the culture dish at 37 ℃, and placing the culture dish at 5% CO₂Culturing in an incubator with saturated humidity.

2.2 subculture of Hair follicle Stem cells

(1) Carrying out enzyme digestion subculture when hair follicle stem cells grow to 60-70% and are fused, (2) discarding cell culture solution when the cultured cells are cultured, adding a proper amount of PBS solution with double antibody, cleaning twice, (3) adding 0.25% trypsin to digest the cells for 1min, (4) adding equal volume of culture solution, stopping digestion, (5) transferring suspension in a dish into a new centrifuge tube, centrifuging at 1500r/min for 5min to precipitate the cells, carefully sucking supernatant, resuspending the cells in the culture solution, and (6) counting the passaged P5 generation hair follicle stem cells by using a cell counting plate method (the number of the cells/m L is equal to the total number of four large cells/4 × 10)⁴One/m L).

2.3 Effect of different concentrations of melatonin stimulation on the proliferative Activity of Hair follicle Stem cells

After the cells are attached to the wall, the liquid is changed every other day, the serum of the stem cell culture medium is changed to exosome-free serum (serum 120000 × g is prepared by overnight centrifugation), a control group (0pg/m L) and a melatonin treatment group (100pg/m L, 300pg/m L, 500pg/m L and 700pg/m L) are set, each concentration is provided with 5 multiple wells, the total volume of each well is 150 mu L, and the cell proliferation activity is tested by adopting an MTT method when the melatonin is added for 1-4 days respectively, wherein the MTT method is specifically as follows:

(1) continuously adding working liquid of 20 mu L MTT (5pg/m L) into each hole, blowing and beating the tip of the tip uniformly, and (2) placing a 96-hole plate at 37 ℃ and 5% CO₂Continuously culturing for 4h in the saturated humidity cell culture box, (3) taking out the culture plate after 4h, sucking out the culture medium in the holes, taking no purple precipitate to the bottom of the plate, (4) adding 200 mu L DMSO into each hole to dissolve the precipitate, placing the 96-well plate in a temperature of 37 ℃ and 5% CO₂Saturated humidity cellIncubating for 30min in an incubator; (5) after 30min, oscillating the 96-well plate for 10min, and testing the OD value by an enzyme-labeling instrument (490 nm); (6) according to the measured OD value, the proliferation of the cells of each group is analyzed and compared, and a cell proliferation activity curve is drawn.

2.4 determination of melatonin concentration

Cell count to adjust cell density 3 × 10⁵One bottle, 25cm in length²The method comprises the steps of adding stem cell culture solution into a cell culture bottle, changing the solution every other day after cell attachment, changing the serum of a stem cell culture medium into exosome-free serum (serum 120000 × g is prepared by overnight centrifugation), setting a control group (0pg/m L) and a melatonin treatment group (100pg/m L, 300pg/m L, 500pg/m L and 700pg/m L), setting 3 repeated culture bottles for each concentration, collecting cell supernatants of each group when melatonin is added for 1-4 days, extracting exosomes of each group by using a kit, and testing the concentration of the exosomes of each group by using a BCA method.

2.4.1 extraction of exosomes

Collecting the culture supernatant of each group, and extracting exosome by a kit method.

(1)300 × g, centrifuging for 10min, 4 ℃, and taking supernatant;

(2)2000 × g, centrifuging for 15min, 4 ℃, and taking supernatant;

(3)10000 × g, centrifuging for 30min, 4 ℃, and taking supernatant;

(4) filtering with 0.22 μm filter, and collecting filtrate;

(5) concentrating by an ultrafiltration tube, and collecting cell supernatant concentrated solution;

(6) transferring the cell supernatant concentrate to a new centrifuge tube and adding 0.5 volume of exosome-separating agent;

(7) mix the media/reagent mixture by vortexing until there is a homogenous solution;

(8) incubating the sample at 2 ℃ to 8 ℃ overnight;

(9) after incubation, the samples were centrifuged at 10,000 × g for 1h at 4 ℃;

(10) the supernatant was discarded, the pellet was exosome and the pellet was resuspended in an appropriate volume of PBS.

2.4.2 determination of exosome concentration

And (3) resuspending the obtained exosome precipitate of each group, adding RIPA lysate for lysis, incubating on ice for 5min, centrifuging at 12000 × g for 5min, taking supernatant, and carrying out Protein quantification by using a BCA method, wherein a BCA Protein Assay Kit (worker) is used in the experiment.

(1) Preparation of standard substance

Diluting 5X solution A with distilled water to obtain 1X solution A, quantitatively taking solution A, wherein solution B is 50:1, according to the total volume of the required BCA working solution, uniformly mixing to obtain BCA working solution, taking 1m L distilled water and 1m L solution D, uniformly mixing to obtain solution E, weighing 10mg of reagent C, dissolving with 0.5m L solution E, and performing vortex oscillation for 60s to obtain solution F.

(2) According to the number of the measured samples, a plurality of holes on the ELISA plate are selected and divided into a standard group and a sample group, wherein 16 holes are provided, 1 repetition is arranged on each standard, a standard protein solution with the corresponding concentration of 5 mu L is respectively added into each hole, and the rest holes are added with samples of 5 mu L of each group and divided into 2 repetition groups.

(3) Add 5. mu. L solution F to each tube and incubate at 37 ℃ for 30 min.

(4) Add 200. mu. L BCA working solution to each well, mix well quickly, incubate for 30min at 37 ℃.

(5) After cooling to room temperature, the a562 value was measured for each well on a microplate reader.

(6) The mean of the absorbance values (562nm) of the wells of the standard set was plotted on a graph paper or in Microsoft Excel software using the ordinate as the mean of the corresponding protein concentration and the abscissa as the mean of the mean.

(7) And calculating the protein concentration of the sample after dilution on a standard curve according to the average value of the same sample, and then calculating the protein concentration of the original sample according to the dilution times.

3 results and analysis

3.1 passage and recovery of Hair follicle Stem cells

After thawing, the hair follicle stem cells are cultured in a culture dish, observed under a microscope and attached to the wall every other day. The hair follicle stem cells grow in an obvious paving stone shape; observed under a high power microscope, the hair follicle stem cells grow in a multinuclear shape, the proportion of cell nucleuses in the cells is larger, and the cells have obvious juvenile cell states.

3.2 melatonin stimulation of Hair follicle Stem cells

3.2.1 morphological Observation of Hair follicle Stem cells

Cells were inoculated into culture plates and attached to the wall every other day. Before melatonin stimulation, the hair follicle stem cells are uniform in shape, and appear to grow like paving stones when being attached to the bottom of the dish, and after melatonin stimulation, the shape of the cells is not obviously changed after 1-2 d. After 3d, the cell proliferation is obviously increased compared with the control group, and the cell morphology is still the hair follicle stem cell morphology and grows in a paving stone-shaped arrangement. At 4d, the cells were seen to have grown over the bottom of the 96-well plate, the nuclear morphology was prominent, and the cells grew more densely.

3.2.2 Effect of different concentrations of melatonin stimulation on the proliferative Activity of Hair follicle Stem cells

The results of MTT method showed that the optimal concentration of melatonin for proliferation of hair follicle stem cells was 500pg/m L, and the light absorption values measured by MTT method were plotted as ordinate to plot the line graph (FIG. 1) showing the effect of different concentrations of melatonin stimulation on the proliferation activity of hair follicle stem cells, as can be seen from the graph, the light absorption values of cells did not change much before 2d of the hair follicle stem cells treated with melatonin, probably because the cells were still adapted to the change of the medium conditions, 3d shows that the light absorption values of the groups of cells were large, and the light absorption values of the melatonin treatment were 0pg/m L, 100pg/m L, 300pg/m L, 500pg/m L, 700pg/m L concentrations of pg 0.1087. + -. 0.0037^a、0.1109±0.0021^ab、0.1236±0.0032^bc、0.1304±0.0059^c、0.1106±0.0057^abIt can be seen that the cell proliferation was significantly increased in the group with melatonin addition (P <0.01) and in the group with melatonin treatment at 500pg/m L the cell proliferation was most significant in the group with melatonin addition at 4d, and the cells continued to proliferate with absorbance values of 0.2428. + -. 0.0077 for melatonin treatment at concentrations of 0pg/m L, 100pg/m L, 300pg/m L, 500pg/m L and 700pg/m L, respectively^a、0.3779±0.0112^e、0.3840±0.0137^e、0.3946±0.0065^e、0.3180±0.0091^cThe cell proliferation of the group with melatonin still obviously increased (P <0.01) compared with the group without melatonin, and the cell proliferation of the group treated by 500pg/m L melatonin is most obviously increasedIn conclusion, melatonin at 500pg/m L was considered to be the optimal concentration for proliferation of hair follicle stem cells.

3.2.3 BCA assay for exosome concentrations

The results of visual differential analysis (fig. 3) were performed on the results of different concentrations of melatonin stimulating the hair follicle stem cells, with the days of cell culture as abscissa and the concentrations as ordinate, showing that the amount of exosome concentration from the hair follicle stem cells increased with the number of days of stimulation after different concentrations of melatonin stimulated the exosome concentration of the hair follicle stem cells increased, the exosome concentration of the hair follicle stem cells increased dramatically with the number of days of stimulation for 3d, the exosome concentration of the 500pg/m L group increased steadily for the other groups, possibly with increasing cell proliferation, 4d stimulation was also increased, the exosome concentration of each group increased pg/m L was more pronounced, but the exosome concentration of the 500pg/m L group reached 4d for 3d, possibly because the cells had proliferated by a greater amount, nearly fully, the cell culture was increased, the exosome concentration of the 500pg/m L group increased to reach the exosome concentration of 4d, the optimal for the BCA treatment was found to be increased in the vial, and the melatonin release was found to be increased in the range 389 concentration range of 300, and 387 was found to be equal to the optimal for the BCA treated group.

TABLE 1 concentration of hair follicle stem cell-derived exosomes after melatonin stimulation (μ g/m L)

Experimental example 2 Effect of melatonin on exosomes derived from hair follicle stem cells

1 test materials and reagents

1.1 Experimental materials

The separated and purified inner Mongolia cashmere goat hair follicle stem cells provided by the laboratory.

1.2 Experimental reagents

DMEM/F12, Fetal Bovine Serum (FBS) from Hyclone, Epidermal Growth Factor (EGF), Melatonin (Meldonin), Tris-base, Glycine from Sigma, L uzindole, 4-P-PDOT from ChemCruz, Insulin-Transferrin-Selenium (ITS), 0.25% trypsin from Gibco, double Antibody (PS), 5 × Protein loading buffer from Sorbox, Total Exosome Isolation Reagent (from Invitrogen), RIPA lysate, BCA Protein Assay Kit, SDS-PAGE denaturing acrylamide gel rapid preparation Kit, SDS, skim milk powder from Shanghai, Mouse Antibody-CD 81 Antibody from Novus, Rabbit Antibody-Antibody Reagent Kit, Western Blot, HSP-Antibody Reagent Kit, HSP-DNA Reagent Kit, HSP-DNA Antibody Reagent Kit, HSP-DNA fragment 387-DNA fragment, HSP-DNA fragment, and DNA fragment.

2 method of experiment

The concentration of melatonin was determined to be 500pg/m L from experimental example 1, and exosomes were extracted 3d after melatonin stimulated cells.

2.1 melatonin stimulation of Hair follicle Stem cells

(1) Cell count to adjust cell density 5 × 10⁵One bottle, insert 75cm²And (4) adding stem cell culture solution into the cell culture bottle for culture.

(2) When the cells grow to 50%, the cells are washed for 2 times by adding an appropriate amount of PBS solution with double antibodies and discarded, and are replaced by a culture solution of exosome-free serum (prepared by 120000 × g of fetal bovine serum by centrifugation overnight).

(3) One group was added with normal stem cell culture medium (containing DMEM/F12, 10% FBS (no exosomes), 1% PS, 20ng/m L EGF, 10 μ L/m L ITS) in an exosome-free serum, and the other group was added with melatonin (MT, 500pg/m L), i.e. (containing DMEM/F12, 10% FBS (no exosomes), 1% PS, 20ng/m L EGF, 10 μ L/m L ITS, 500pg/m L MT).

(4) After 3 days of culture, two groups of cell supernatants were collected, respectively.

2.2. Melatonin stimulation (receptor blocking) of hair follicle stem cells

The method is the same as experiment 2.1, and melatonin receptor blocker 500pg/m L melatonin +500pg/m L L uzindole (L uz, MTI + MT2 receptor blocking), 500pg/m L melatonin +500pg/m L4-P-PDOT (4-P, MT2 receptor blocking) is selected to stimulate the hair follicle stem cells.

2.3 extraction of exosomes

The same as 2.4.1 of experimental example 1.

2.4 determination of exosome concentration

The same as 2.4.2 of experimental example 1.

2.5 identification of exosomes derived from Hair follicle Stem cells

2.5.1 Transmission Electron microscopy identification of exosome morphology from Hair follicle Stem cells

Diluting the separated exosome suspension 10 mu L with an equal volume of PBS solution, then dropwise adding the exosome suspension on a sample-carrying copper net with the diameter of 2mm, standing at room temperature for 1min, lightly sucking out excess liquid by using filter paper, counter-dyeing with 3% sodium phosphotungstate solution (pH6.8) at room temperature for 5min, double-steaming and washing once, airing at room temperature for 2min, observing each visual field area by using a transmission electron microscope, randomly selecting exosomes, measuring the diameters of the exosomes and taking pictures.

2.5.2Western blot to detect the expression levels of exosome marker proteins HSP70, CD9 and CD81

(1) Preparing separation gel: cleaning the glass sheet with SDS-PAGE gel and comb, air drying, mounting on the gel plate, injecting deionized water into the hole along the edge hole of glass, waiting for 20min to check if water leaks, and reinstalling if water leaks. Preparing 12% separation glue according to the formula, quickly mixing uniformly, adding the mixture to the position slightly below the separation position of the concentrated glue of the glass plate, removing air bubbles, and injecting deionized water above the separation glue.

(2) Preparing concentrated glue: after 30min, preparing concentrated glue after the separation glue is solidified, quickly mixing uniformly, pouring out the deionized water above the separation glue, adding the concentrated glue, immediately inserting a comb, and waiting for the glue to be solidified.

(3) Sample application: and after the rubber plate is well prepared, putting the rubber plate into an electrophoresis tank, introducing SDS electrophoresis buffer solution into the electrophoresis tank to scale marks, pulling out a comb, and introducing a sample into each point sample hole along the wall.

(4) Electrophoresis: after the sample application is finished, the cover is covered, and concentrated glue is firstly run by using the voltage of 120V, and separation glue is run by using 80V instead.

(5) When the protein runs below the gel 2/3, the electrophoresis apparatus is closed, and the electrophoresis buffer is recovered and used for the next experiment.

(6) Film transfer (wet transfer): cutting a PVDF membrane (the edge of the gel can be cut off) with the same size as the gel, soaking in methanol for 1-2min, taking out with forceps, and soaking in membrane-transferring buffer solution for 1-2min, wherein the time is not too long, otherwise the concentration of protein bands is affected. The film-rotating clamp is opened, and the negative electrode (black), the sponge, the filter paper, the glue, the film, the filter paper, the sponge and the positive electrode (white) are placed in sequence. And clamping the black-white clamp, and putting the black-white clamp into a film rotating device. Pouring pre-cooled membrane conversion buffer solution into the membrane conversion tank, putting an ice bag with a proper size into a compartment in the membrane conversion tank, covering a cover, and keeping constant current for 350mA for 90 min.

(7) Blocking, preparing 5% skimmed milk powder with 1 × TBS, gently taking out the membrane, placing in blocking solution, placing in shaking table, and standing at room temperature for 2 hr.

(8) Primary antibody hybridization: after blocking was complete, the blocking solution was recovered and the membrane was washed four times 5min each with TBST. Primary antibody was diluted with TBST as required by the instructions, and after all air bubbles were removed, the mixture was allowed to stand at 4 ℃ overnight for hybridization.

(9) And (3) hybridization of a second antibody: the primary antibody was recovered and the membrane washed with TBST four times for 5min each. The secondary antibody was diluted with TBST and hybridized for 1h at room temperature as per the instructions.

(10) Imaging: after hybridization of the secondary antibody, the secondary antibody diluent was discarded, and the membrane was washed with TBST four times for 5min and TBS once for 5 min. And detecting the protein expression content by using an Odyssye infrared laser scanning imaging system and then analyzing.

3 results and analysis

3.1 culture of Hair follicle Stem cells

After 2d, the cells grow to 50 percent, and are replaced by a culture medium containing melatonin, melatonin (receptor blocking) L uzindole (L uz) and 4-P-PDOT (4-P). before the melatonin stimulates, the hair follicle stem cells have uniform shape and appear paving stone growth on the whole body attached to the bottom of a dish, and after the melatonin and the melatonin (receptor blocking) are added for stimulation, the cell shape of a treatment group is not obviously changed compared with that of a control group, but the number of the cells of the melatonin stimulation group is increased.

3.2 determination of exosome concentration

Analyzing the concentrations of exosomes derived from the hair follicle stem cells of different treatment stimuli (see table 2), and visually and differentially analyzing the results by taking the cell culture days as the abscissa and the concentrations as the ordinate (see fig. 4), wherein the exosomes derived from the hair follicle stem cells of the group without melatonin treatment are named as CG-Exo, the exosomes derived from the hair follicle stem cells of the group with 500pg/m L melatonin treatment are named as MT-Exo, the exosomes derived from the hair follicle stem cells of the group with 500pg/m L melatonin (receptor blocker L uzindole) are named as L uz-Exo, the exosomes derived from the hair follicle stem cells of the group with 500pg/m L melatonin (receptor blocker 4-pgP-PDOT) are named as 4-P-Exo, and the results show that the concentrations of exosomes derived from the hair follicle stem cells of each group are 939.33 +/-6.61.61 through 939.33 +/-6.61 respectively^a、1352.50±9.36^e、1053.33±8.57^c、958.83±8.49^aThe exosome concentration was significantly increased relative to the control group after melatonin addition, possibly related to the sustained proliferation of cells, and the exosome concentration was decreased after two groups of melatonin receptor blockers were added, possibly because the receptors blocked melatonin receptors and the cells returned to their normal unstimulated state, so the 4-P-Exo group differed little from the CG-Exo group, whereas the L uz-Exo group differed slightly from the CG-Exo group, possibly because the two receptor blockers blocked receptors, the L uz group was slightly higher than the 4-P treated group, L uz mainly blocked melatonin receptors 1 and 2, 4-P mainly blocked melatonin receptor 2, and in the case of melatonin membrane receptor blocker addition, the exosome decreased in both groups, so it was presumed that in this experimental condition, it was achieved by the control of the melanin membrane receptor 2, the same condition was not exerted, so that the MT-exosomal function was not consistent, and the primary difference was considered to be a significant increase in the concentration of dry hair follicles after the three groups had been treated with melatonin.

TABLE 2 concentration of exosomes derived from the hair follicle stem cells (μ g/m L) for each group

Note: the same letters in the same row of data in the table indicate no significant difference (p >0.05), adjacent letters significantly (p <0.05), and alternate letters significantly (p < 0.01).

3.3 identification of exosomes

3.3.1 Transmission Electron microscopy identification of exosome morphology from Hair follicle Stem cell sources

Most exosomes are slightly smaller than 100nm in the field of an electron microscope, and meanwhile, the exosomes have a structure like a saucer or a concave hemisphere, have an obvious membrane and a clear three-dimensional structure, and the result shows that the exosomes derived from hair follicle stem cells after stimulation of the hair follicle stem cells and melatonin all present a typical exosome structure (see fig. 5).

3.3.2 Western blot to detect the expression levels of exosome marker proteins HSP70, CD9 and CD81

CG-Exo, MT-Exo, L uz-Exo and 4-P-Exo are respectively subjected to repeated sample loading for 3 times, compared with CG-Exo, L uz-Exo and 4-P-Exo, the expression quantity of MT-Exo protein is obviously up-regulated, the difference of the protein expression quantity among CG-Exo, L uz-Exo and 4-P-Exo is not obvious, and the result is consistent with the quantitative result of BCA protein, and the extracted exosome is effective (shown in figure 6), wherein HSP70 is heat shock protein which has the best expression abundance, CD9 and CD81 are also expressed, CD81 bands are dispersed, possibly related to sample species, and the capacities of the expressed protein are different among different species.

Experimental example 3 Effect of exosomes derived from Hair follicle Stem cells on receptor cells

1 test materials and reagents

1.1 Experimental materials

The inner Mongolia white cashmere goat P5 generation hair follicle stem cell comes from exosome, hair follicle stem cell and hair papilla cell.

1.2 Experimental reagents

DMEM/F12, Fetal Bovine Serum (FBS) from Hyclone, Epidermal Growth Factor (EGF), Melatonin (Meldonin), PKH67, crystal violet from Sigma, L uzindole, 4-P-PDOT from ChemCruz, Insulin-Transferrin-Selenium (ITS), 0.25% trypsin from Gibco, bis-antibody (PS), 4% paraformaldehyde, Triton X100, DAPI from Sorbox, Total oxygen Isolation Reagent (BCA) from Invitrogen, BCA Protein Assay Kit, BSA from Shanghai, RNAiso Plus, reverse transcription Kit, PCR, 2 × SYBR Yeast Ex Taq from TAKARA.

2 method of experiment

2.1 Hair follicle Stem cells and Hair papilla cell uptake exosomes

2.1.1 PKH 67-labeled exosomes

PKH67 stably binds to the lipid bilayer of the cell membrane and is a green fluorescent cell labeling reagent dye that is often used to label exosomes containing lipid bilayer membranes. The method comprises the following specific steps:

(1) exosomes were resuspended in Diluent C in 1m L kit;

(2) adding a Diluent C Diluent in a 1m L kit into a 4 mu L PKH67 dye solution for dilution;

(3) uniformly mixing the solution (1) and the solution (2), incubating at room temperature for 2-5 min, and frequently and slightly inverting the centrifuge tube to fully mix the solutions;

(4) adding 1% BSA with the same amount for incubation for 1min, and stopping the staining reaction;

(5)100000 × g, centrifuging at 4 deg.C for 70min, and removing supernatant;

(6) PBS resuspended exosome pellet.

2.1.2 fluorescent microscope Observation of Hair follicle Stem cells and Hair papilla cells uptake into exosomes

(1) Inoculating the P5 generation hair follicle stem cells and hair papilla cells into a 12-pore plate paved with 22 × 22mm cell slide;

(2) after the cells adhere to the wall, adding a hair follicle stem cell source exosome marked by PHK67 dye, and incubating for 12 h;

(3) after 12h, the cells were washed 3 times with PBS;

(4) fixing cells with 4% paraformaldehyde for 30 min;

(5) washing with PBS for 3 times, each for 3 min;

(6) staining nuclei with 1 μ g/m L DAPI at room temperature for 15 min;

(7) washing with PBS for 3 times, each for 3min, and removing excessive dye;

(8) finally, the treated cells are placed on a glass slide for mounting, and the uptake condition of hair follicle stem cells and hair papilla cells to exosomes is analyzed by a fluorescence microscope.

2.2 Effect of Hair follicle Stem cell-derived exosomes on Hair follicle Stem cell and Hair papilla cell proliferation

The cell counting is to adjust the cell density to 2000 per well, the cells are connected into a 96-well plate, the stem cell culture solution is added into hair follicle stem cells, the hair papilla cell culture solution is added into the hair follicle stem cells, the solution is changed after the cells are attached to the wall, the serum of the culture solution is changed into serum without exosome (prepared by serum 120000 × g through overnight centrifugation), the experiment is divided into a control group and an exosome treatment group, the cell culture solution is added into each group, the exosome treatment group comprises CG-Exo, MT-Exo, L uz-Exo and 4-P-Exo, three concentration gradients of 100 mu g/m L, 300 mu g/m L and 500 mu g/m L are arranged in each exosome treatment group, 3 multiple wells are arranged for each concentration, the total volume of each well is 150 mu L, and the proliferation activity condition of the receptor cells is tested by adopting an MTT method when exosome is added for 1-4 days.

2.3 Effect of Hair follicle Stem cell-derived exosomes on receptor cell migration

Suspending cells in serum-free medium, adjusting cell density to 1 × 10⁴The experiment is divided into a control group and an exosome treatment group, wherein the exosome treatment group comprises four exosome treatments of CG-Exo, MT-Exo, L uz-Exo and 4-P-Exo, and three concentration gradients of 100 mu g/m L, 300 mu g/m L and 500 mu g/m L are set in each exosome treatment group, and a trans well chamber of corning company is adopted in the migration experiment, and the specific method is as follows:

(1) the method comprises the steps of (1) inoculating 200 mu L cell suspension in a Transwell upper chamber, adding 500 mu L of an exosome-free serum culture medium and PBS into a control group lower chamber, adding 500 mu L of an exosome-free serum culture medium and exosomes with different concentrations into an experimental group lower chamber, (2) terminating culture after 48 hours (the hair papilla cells are 24 hours), discarding the culture medium, slightly wiping residual cells in the upper chamber with a sterile cotton swab, (3) slightly cleaning the cells with PBS, fixing with 4% paraformaldehyde for 20min, discarding the fixing solution, cleaning with distilled water for 2 times, staining with 1% crystal violet for 20min, discarding the staining solution, cleaning with distilled water for 2 times, observing under an inverted phase-contrast microscope, counting transmembrane migration cells in 6 random fields per group, taking the average value, and repeating the experiment for three times.

2.4 Effect of Hair follicle Stem cell-derived exosomes on receptor cell Hair follicle development-related genes

Recipient cells were plated into 24-well plates, 5% CO, grouped according to experiment 2.3₂Culturing in saturated humidity incubator, and extracting total RNA of cells of each treatment group respectively at 1 day, 3 days, 5 days, 7 days, 14 days and 21 days after adding exosome for detecting expression quantity of each gene in the development process of receptor cells.

2.4.1 extraction and identification of Total RNA from receptor cells

RNAioso Plus lyses each group of treated recipient cells, then extracts total RNA, determines RNA concentration and OD₂₆₀/OD₂₈₀Ratio, integrity determination by 1% agarose gel electrophoresis.

2.4.2 RT-PCR primer design and Synthesis

Primers were designed using the Priemer 5.0 software according to the goat gene sequences published in GenBank.

2.4.3 RT-PCR amplification

2.4.4 relative quantitative PCR detection of expression between different groups of genes

2.5 statistical analysis of the Experimental data

The gene expression abundance adopts (1+ E) of a target gene and an internal standard gene^-△△CtWherein E is the amplification efficiency, and 2 is used when the amplification efficiency of the target gene and the housekeeping gene is close to 100%^-△△CtAnd (4) showing.

3 analysis of results

3.1 Hair follicle Stem cells and Hair papilla cell uptake exosomes

Each set of exosomes labeled with cell membrane marker PKH67 was added to recipient cells hair follicle stem cells and hair papilla cells, respectively, for 15 hours of incubation. And (3) detecting by a confocal microscope, wherein each group of exosomes can be taken up by recipient cells hair follicle stem cells and hair papilla cells, and each group of exosomes can be absorbed by the recipient cells as shown by the fact that the exosomes (green) marked by PKH67 enter the recipient cells and surround the cell nucleus (blue) of the recipient cells.

3.2 Effect of Hair follicle Stem cell-derived exosomes on recipient cell proliferation

3.2.1 Effect of Hair follicle Stem cell-derived exosomes on Hair follicle Stem cell proliferation

The results show that the addition of exosomes increases the proliferation of hair follicle stem cells significantly and that the addition of exosomes L uz-ecto and 4-P-Exo to the melanin-treated hair follicle stem cells can significantly promote the increase of stem cells while the addition of exosomes L-Exo, 4-Exo and exosomes exp (receptor blocking) to the melanin-treated hair follicle stem cells can significantly increase the proliferation of hair follicle stem cells, and that the increase of the proliferation of hair follicle stem cells is more significant when the skin follicle stem cells are treated with exosomes of different groups (CG-Exo, MT-Exo is treated with no treatment) than when the skin follicle stem cells are treated with no treatment (fig. 7), and that the skin-like hair follicle stem cells are treated with exosomes of different concentrations (CG-Exo, MT-Exo and 4-Exo) are treated with melatonin (receptor blocking) than when the skin-like hair follicle stem cells are treated with no treatment (fig. 3), the skin-Exo and the skin-Exo-exosomes of the melanin-exosomes are treated with melatonin (receptor blocking) and the skin cells can significantly increase the skin-Exo and the skin-Exo-exosomes can significantly promote the skin proliferation of hair follicle stem cells after the skin cells are treated with high skin cells.

TABLE 3100 μ g/m L Effect of Hair follicle Stem cell-derived exosomes on Hair follicle Stem cell proliferation

TABLE 4300. mu.g/m L Effect of Hair follicle Stem cell-derived exosomes on Hair follicle Stem cell proliferation

TABLE 5500 μ g/m L Effect of hair follicle stem cell-derived exosomes on hair follicle stem cell proliferation

3.2.2 Effect of Hair follicle Stem cell-derived exosomes on Hair papilla cell proliferation

Similar to the addition of hair follicle stem cells to each group of exosomes, the proliferation of hair follicle papilla cells increased after the addition of exosomes (see fig. 8). the melatonin-treated hair follicle stem cell-derived exosomes MT-Exo significantly promoted the increase of hair follicle stem cells compared to untreated hair follicle stem cell-derived exosomes CG-Exo, and melatonin (receptor blocking) -treated exosomes L uz-Exo, 4-P-Exo, similarly, the differences between L uz-Exo, 4-P-Exo and CG-Exo were smaller, but for hair follicle cells, the different concentrations of the same exosomes added appeared not to be different on the first 2 days, particularly at concentrations of 300 μ g/m L and 500 μ g/m L, but overall, the stem cell-derived exosomes were able to promote the proliferation of hair follicle cells, while the melanin-treated hair follicle stem cell-derived exosomes promoted the proliferation of hair follicles more significantly (see table 6, table 7).

TABLE 6100 μ g/m L Effect of Hair follicle Stem cell-derived exosomes on Hair papilla cell proliferation

TABLE 7300 μ g/m L Effect of Hair follicle Stem cell-derived exosomes on Hair papilla cell proliferation

TABLE 8500 μ g/m L Effect of Hair follicle Stem cell-derived exosomes on Hair papilla cell proliferation

3.3 Effect of Hair follicle Stem cell-derived exosomes on receptor cell migration

3.3.1 Effect of Hair follicle Stem cell-derived exosomes on Hair follicle Stem cell migration

The results of Transwell migration experiments showed that the group to which melatonin was added promoted the migration of hair follicle stem cells compared to the control group (see fig. 9). when exosomes derived from hair follicle stem cells were added, the migration effect of cells was rapidly increased at the same time, the exosomes MT-Exo derived from hair follicle stem cells after melatonin treatment significantly promoted the increase of hair follicle stem cells compared to the exosomes CG-Exo derived from untreated hair follicle stem cells and the exosomes L uz-Exo, 4-P-Exo treated with melatonin (receptor blocking).

3.3.2 Effect of Hair follicle Stem cell-derived exosomes on Hair papilla migration

Similarly to the migration of hair follicle stem cells, the melatonin-stimulated group promoted the migration of hair papilla cells compared to the control group (see fig. 10). similarly, the migration effect of hair follicle stem cell-derived exosomes was rapidly increased, and the melatonin-treated hair follicle stem cell-derived exosomes MT-Exo significantly promoted the increase of hair papilla cells compared to untreated hair follicle stem cell-derived exosomes CG-Exo, and melatonin (receptor-blocking) treated exosomes L uz-Exo and 4-P-Exo.

3.3.3 extraction of Total RNA from cells

Total RNA of P5 generation hair follicle stem cells and hair papilla cells after treatment of the control group and the experimental group is extracted by using RNAioso Plus, and the detection result of 1% agarose gel electrophoresis shows that the 28S and 18S bands are clearer. The RNA has better integrity and basically has no obvious degradation, and is suitable for subsequent experiments.

3.4 Effect of Hair follicle Stem cell-derived exosomes on receptor cell Hair follicle development-related genes

3.4.1 Effect of Hair follicle Stem cell-derived exosomes on Hair follicle development-associated genes β 1-integrin, SOX9, FGF5, β -catenin, KAP7, KAP8 of Hair follicle Stem cell

β -integrin and SOX9 are stem cell specific expression genes, and after melatonin and melatonin treated exosomes are added to hair follicle stem cells, the expression level of the cells is gradually increased at 0-5d, and the expression level of the sterny genes is reduced after 5 d.FGF 5 is a wool growth-inhibiting gene which mainly regulates the periodic development of hair follicles, and is expressed during the growth of hair follicles, and reaches the maximum at 3d, and then is reduced at 5 d. β -catenin has a great effect as an exogenous factor for regulating the differentiation of hair follicle stem cells as a key factor of Wnt signal pathway.A high activation of β -catenin can lead hair follicle stem cells to differentiate towards hair follicle cells, and lack or low-intensity activation of β -catenin promotes the differentiation into cells in the epidermal direction.A gradual increase of the expression level at 0-3d, a subsequent decrease of the expression level after 7d, but a further increase of the expression level after 7d, possibly after stem cells differentiate into hair follicle cells, a further increase of the signal pathway of KAP 63-7 and KAP 78, and a marked increase of hair follicle stem cells after 7d, and when the differentiation of hair follicle stem cells are obviously increased (see figure 6711, 6711).

3.4.2 influence of exosome derived from hair follicle stem cells on hair follicle development related genes FGF5, β -catenin of hair papilla cells

Like hair follicle stem cells, FGF5 expression level in hair papilla cells reaches maximum at 3d, and then expression level is reduced β -catenin is a key factor of Wnt signaling pathway to regulate growth of hair papilla cells, reaches maximum at 1d, and then expression level is reduced.

Claims

1. Use of exosomes derived from hair follicle stem cells for promoting differentiation of hair follicle stem cells into hair follicle cells.

2. Use of exosomes derived from hair follicle stem cells in promoting hair follicle stem cell proliferation.

3. Use of exosomes derived from hair follicle stem cells for promoting hair papilla cell proliferation.

4. A method of promoting differentiation of hair follicle stem cells into hair follicle cells, comprising: adding hair follicle stem cell-derived exosomes to the proliferated hair follicle stem cells.

5. The method according to claim 4, wherein said hair follicle stem cell-derived exosomes are prepared by a method comprising: adding melatonin to hair follicle stem cells promotes secretion of exosomes from hair follicle stem cells.

6. A method of promoting secretion of exosomes from hair follicle stem cells, comprising: melatonin was used to stimulate hair follicle stem cells to promote exosome secretion.

7. The method as claimed in claim 6, wherein the concentration of melatonin added is 100-700pg/m L.

8. The method according to claim 7, wherein melatonin is added at a concentration of 500pg/m L.

9. A method of promoting proliferation of dermal papilla cells, comprising: the hair papilla cell is added with exosome secreted by hair follicle stem cells to promote the proliferation of the hair papilla cells.

10. The method according to claim 9, wherein in preparing exosomes secreted from hair follicle stem cells, melatonin is added to the hair follicle stem cells to promote secretion of exosomes from hair follicle stem cells.